The mechanism of stimulus response coupling mediated by Ca2+ and calmodulin was studied at two levels: 1) We demonstrated that the cooperative binding of calcium to calmodulin, leading to a stepwise conformational change, is due to an allosteric transition affecting the amino and carboxyl-terminal halves of the molecule. Binding of two mol of Ca2+ decreases the affinity for the two remaining sites. The central helix which connects the two halves of the molecule plays a critical role in this negative cooperativity. Mutant calmodulins with altered Ca2+-binding properties are being studied to elucidate the role of this structural change in calmodulin function. 2) Studies of the interaction of calmodulin with calcineurin, a calmodulin-stimulated protein phosphatase, have included the isolation and sequencing of the cDNA clones of the two subunits of the enzyme. The complete human cDNA clone of the small subunit was isolated and sequenced. The mapping and amino acid sequence of three of the functional domains of the large subunit (catalytic site, calmodulin-binding and inhibitory domains) was affected by protein sequencing. The sequence data are being used to aid the isolation and complete sequencing of the human calcineurin A gene.